Johne's disease is a chronic, debilitating disease of ruminants (e.g. cows) characterized by diarrhea which usually leads to death. Infected cattle may not exhibit debilitating symptoms for a long period after infection. By the time the symptoms are evident, three to four years of investment in the cattle has been wasted. Further, over that period other cattle and pasture areas can be exposed to the disease. A test that could be performed within a week's time, that is relatively inexpensive to perform, that would be specific for Johne's disease, and that would be sensitive enough to detect the disease in its early stages using fecal samples, would be a useful tool in herd management, regulatory medicine, and other aspects of work with Johne's disease.
The art has previously developed DNA hybridization probes for the identification of some other Mycobacterium (e.g. leprae and bovis). The hybridization technique depends upon finding DNA sequences from the bacteria that will stick (hybridize) to the bacterial DNA, are short enough to be cloned, and are unique enough not to hybridize to DNA from other bacteria. One then labels the probe with a colorimetric or radioactive marker, and then uses the probe to locate the DNA in a sample.
Because of the high degree of homology between M. bovis, M. phlei and M. scrofulaceum on the one hand and M. paratuberculosis on the other, and the presence of M. bovis, M. phlei, and/or M. scrofulaceum in some bovine fecal samples, DNA probe hybridization techniques did not appear to be well suited to be used in a Johne's test (because of the lack of specificity of most DNA sequences in these bacteria). Moreover, hybridizing Mycobacterium DNA has in the past been hindered by the laborious procedures required to break open the bacterial cell to obtain access to the cell DNA (in order to permit use of hybridization techniques). In this regard, M. paratuberculosis has a very thick lipid-rich cell wall. This, and slow replication of this mycobacteria, can make use of conventional chemical cell wall disruption techniques especially tedious. For example, weakening bacterial cell walls by growth for several generations in the presence of 100 to 200 mM glycine, and lysis achieved by lysozyme, proteinase K, and sodium dodecyl sulfate treatment has been tried. However, such procedures can take days.
Conventional mechanical lysis procedures, such as the French press, the Hughes press, and the Ribi pressure cell, take less time than chemical methods (minutes compared with hours or days). They use pressure for rupture of the bacteria. Briefly, bacteria are placed in a chamber where a piston compresses the atmosphere of the chamber (80,000 lbs./square inch). The pressure is then released through a small super cooled valve and the sudden pressure change ruptures the cells. However, growing the cell cultures and achieving the appropriate concentration and volume can be time-consuming. In addition, impurities, such as those that might be present in feces, can damage the equipment used in these techniques.